Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella

Abstract

Comparison of 18S rDNA gene sequences is a very promising method for identification and classification of living organisms. Molecular identification and discrimination of different Dunaliella species were carried out based on the size of 18S rDNA gene and, number and position of introns in the gene. Three types of 18S rDNA structure have already been reported: the gene with a size of ~1770 bp lacking any intron, with a size of ~2170 bp consisting one intron near 5' terminus, and with a size of ~2570 bp harbouring two introns near 5' and 3' termini. Hereby, we report a new 18S rDNA gene arrangement in terms of intron localization and nucleotide sequence in a Dunaliella isolated from Iranian salt lakes (ABRIINW-M1/2). PCR amplification with genus-specific primers resulted in production of a ~2170 bp DNA band, which is similar to that of D. salina 18S rDNA gene containing only one intron near 5' terminus. Whilst, sequence composition of the gene revealed the lack of any intron near 5' terminus in our isolate. Furthermore, another alteration was observed due to the presence of a 440 bp DNA fragment near 3' terminus. Accordingly, 18S rDNA gene of the isolate is clearly different from those of D. salina and any other Dunaliella species reported so far. Moreover, analysis of ITS region sequence showed the diversity of this region compared to the previously reported species. 18S rDNA and ITS sequences of our isolate were submitted with accesion numbers of EU678868 and EU927373 in NCBI database, respectively. The optimum growth rate of this isolate occured at the salinity level of 1 M NaCl. The maximum carotenoid content under stress condition of intense light (400 mumol photon m-2 s-1), high salinity (4 M NaCl) and deficiency of nitrate and phosphate nutritions reached to 240 ng/cell after 15 days.

Figure 1

Amplification with conserved primers of MA1-MA2. The PCR product was analyzed by electrophoresis using 1% agarose gel and marked using 1 kb DNA ladder (Fermentas Co.).

Figure 2

18S rDNA sequence alignment of Dunaliella sp. ABRIINW-M1/2 and other Dunaliella species. Data for other species were gathered from NCBI. The conserved regions of the gene are demonstrated in different colour (red).

Figure 3

Diagram showing the position of intron insertions in 18S rDNA gene of Dunaliella species. 18S rDNA arrangement is demonstrated in 5 group based on the insertion site of introns. The names of the species wich have the corresponding type of arrangement is written in left side.

Figure 4

Phylogram based on 18S rDNA gene for the species of Dunaliella and Dunaliella sp. ABRIINW M1/2. The tree is constructed using neighbor-joining method. Bootstrap values were calculated from 1000 replicates.

Figure 5

18S rDNA intron alignment of Dunaliella sp. ABRIINW-M1/2 and other Dunaliella species. Data for other species were gathered from NCBI. The conserved regions of the gene are demonstrated in different colour (red).

Figure 6

Phylogram based on the intron of 18S rDNA gene for the species of Dunaliella and Dunaliella sp. ABRIINW M1/2. The first and second introns of the species D. parva and D. bardawil are represented with 1 and 2, respectively. The tree is constructed using neighbor-joining method. Bootstrap values were calculated from 1000.

Figure 7

ITS amplification with the primers of AB1-AB2. The PCR product was analyzed by electrophoresis using 1% agarose gel and marked using 1 kb DNA ladder (Fermentas Co.).

Figure 8

ITS (ITS1, 5.8 rDNA and ITS2) sequence alignment of Dunaliella sp. ABRIINW-M1/2 and other Dunaliella species. Data for other species were gathered from NCBI.

Figure 9

Dendrogram showing the relationship among Dunaliella sp. ABRIINW-M1/2 and species of Dunaliella. The tree is based on ITS region and was constructed using the neighbor-joining method. Bootstrap values were calculated from 1000.